[The Study of Recombinant Interfering Lentiviruses and Overexpressed Adenovirus Vectors Targeting Human c-Cbl Gene： Construction, Identification and Efficacy].

OBJECTIVE: To construct recombinant lentivirus and adenovirus which regulate the expression of c-Cbl gene and evaluate their efficacy. METHODS: The interference lentivirus and overexpressed adenovirus targeting human c-Cbl gene were constructed by gene recombination technology. Quantitative PCR and western blotting were used to detect the expression changes in c-Cbl gene and its transcription after leukemia cells (HL60,THP1) were infected by virus. RESULTS: Three recombinant interfering lentiviral vectors targeting human c-Cbl genes to successfully constructed and were identified by DNA sequencing, and the titers of the packaged viruses were all greater than 1×108 TU/ml. Among them, shRNA-2 lentivirus had the highest interference efficiency, and the expression of c-Cbl gene and CBL protein were decreased about 95% and 60% respectively after leukemia cells were infected with shRNA-2; In addition, the recombinant overexpression adenovirus targeting human c-Cbl gene was packaged successfully with the virus titer greater than 1×109 TU/ml. When leukemia cells were infected with adenovirus, the expression of c-Cbl gene and CBL protein were up-regulated about 10 times and 1.5 times respectively. CONCLUSION: Both recombinant interfering lentivirus and overexpression adenovirus can efficiently infect leukemia cells and affect the expressions of c-Cbl gene and CBL protein. It will lay a preliminary foundation for the subsequent study on the function of c-Cbl gene in tumor cells.

Distinct serotonergic pathways to the amygdala underlie separate behavioral features of anxiety.

Anxiety-like behaviors in mice include social avoidance and avoidance of bright spaces. Whether these features are distinctly regulated is unclear. We demonstrate that in mice, social and anxiogenic stimuli, respectively, increase and decrease serotonin (5-HT) levels in basal amygdala (BA). In dorsal raphe nucleus (DRN), 5-HT∩vGluT3 neurons projecting to BA parvalbumin (DRN5-HT∩vGluT3-BAPV) and pyramidal (DRN5-HT∩vGluT3-BAPyr) neurons have distinct intrinsic properties and gene expression and respond to anxiogenic and social stimuli, respectively. Activation of DRN5-HT∩vGluT3→BAPV inhibits 5-HT release via GABAB receptors on serotonergic terminals in BA, inducing social avoidance and avoidance of bright spaces. Activation of DRN5-HT∩vGluT3→BA neurons inhibits two subsets of BAPyr neurons via 5-HT1A receptors (HTR1A) and 5-HT1B receptors (HTR1B). Pharmacological inhibition of HTR1A and HTR1B in BA induces avoidance of bright spaces and social avoidance, respectively. These findings highlight the functional significance of heterogenic inputs from DRN to BA subpopulations in the regulation of separate anxiety-related behaviors.

Chronic administration of Liu Wei Dihuang protects rat's brain against D-galactose-induced impairment of cholinergic system.

This study was aimed to investigate the protective effect of Liu Wei Dihuang (LWDH) against D-galactose (D-gal)-induced brain injury in rats and the existence of sex-dependent differences in LWDH protection. Sixty-four rats evenly composed of males and females were randomly assigned into 4 groups (n = 8): normal saline (NS) + NS (N + N), NS + LWDH (N + L), D-gal + NS (D + N) and D-gal + LWDH (D + L) groups. Rats in D + N and D + L groups received daily injection of D-gal (100 mg/kg, s.c.) for six weeks to establish the aging model, while rats in N + N and N + L groups were injected with the same volume of NS. From the third week, rats in N + L and D + L groups were orally administered with a decoction of LWDH for subsequent six weeks. Rats in N + N and D + N groups were orally administered just with the same volume of NS simultaneously. Morris water maze test was employed to evaluate the ability of learning and memory of the rats in all the groups. Acetylcholine (ACh) content, activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in visual cortex were assayed. Hematoxylin and eosin (HE) staining were used to observe the morphologic injury in hippocampus and visual cortex, and immunohistochemistry was performed to evaluate ChAT and AChE expression levels in the visual cortex. The results showed that the rats in D + N groups exhibited a longer escape latency to platform, lower swimming speed, less percent of target quadrant search time and platform crossings, compared with N + N groups, suggesting the establishment of aging model, while LWDH improved these indexes in D-gal-treated rats. Compared with D + N groups, LWDH increased ACh content and ChAT activity, and decreased AChE activity in visual cortex. Remarkable loss of neurons was found in hippocampus and visual cortex of aging rats, and the injury was significantly attenuated by LWDH. Immunohistochemistry showed D-gal-induced decreases of ChAT and AChE expressions were restored by LWDH. Furthermore, under the neural protection of LWDH, the improvement on platform crossings in male aging rats was better than that in female ones, while in ChAT expression and neuron density in visual cortex, female aging rats obtained more amelioration. These results suggest LWDH can markedly reverse the D-gal-induced cognitive impairments and neuronal damage in both hippocampus and visual cortex, which are achieved at least partly through restoring cholinergic system in central nervous system. Moreover, there is some sex difference in protective effects of LWDH against D-gal-induced impairment.

Comparison of gene mutation spectra in younger and older Chinese acute myeloid leukemia patients and its prognostic value.

Differences in the gene mutation spectra of younger and older Chinese adult AML patients and the prognostic significance of these differentially presented gene mutations are rarely reported. One hundred and thirteen newly diagnosed Chinese adults with AML, divided into groups of younger and older patients, were enrolled in this study. Bone marrow samples from the patients were analyzed using targeted next-generation sequencing with a panel of 141 genes. Ninety-eight mutated genes were detected and the top 10 mutated genes were KMT2D, FLT3, FAT1, ASXL1, NRAS, DNMT3A, RELN, TET2, JAK2, and KRAS. The top five functional groups were the tyrosine kinase pathway, transcription factors, DNA methylation, chromatin modifiers, and the JAK-STAT signaling pathway. Younger patients exhibited higher incidences of KMT2D (33.8% vs 10.4%, P = 0.004) and KRAS (15.4% vs 2.1%, P = 0.042) mutations than older patients; whereas, older patients harbored more SRSF2 (20.8% vs 0%, P = 0.002), transcription factor (85.4% vs 67.7%, P = 0.031), DNA methylation (58.3% vs 36.9%, P = 0.024), and RNA splicing (31.3% vs 12.3%, P = 0.013) mutations than younger patients. Moreover, patients with SRSF2 mutations exhibited a lower rate of overall survival (P < 0.001) and relapse-free survival (P < 0.001) than patients carrying wild-type SRSF2. In conclusion, rarely reported KMT2D, FAT1, and RELN mutations were detected at high frequencies in our cohort. The gene mutation spectrum of older patients was different to that of younger patients. Moreover, older patients harbored more SRSF2 mutations, which predicted lower rates of overall and relapse-free survival.

Publisher Correction: Cannabinoid CB1 receptors in the amygdalar cholecystokinin glutamatergic afferents to nucleus accumbens modulate depressive-like behavior.

In the version of this article originally published, there were several errors in Fig. 4. In Fig. 4a, the title read '3D repeated optical inhibition after CSDS.' It should have read '3-day repeated optical inhibition after CSDS.' In Fig. 4c, two labels that should have been aligned with the time axis appeared in the wrong place in the figure. The ticks labeled 'SI' and 'Fiber implant' should have also been labeled with '10' and '14,' respectively. Additionally, in Fig. 4j, a label that should have been aligned with the time axis appeared in the wrong place in the figure. The tick labeled 'Fiber implant' should have also been labeled with '14.' The errors have been corrected in the print, PDF and HTML versions of the manuscript.

Cannabinoid CB1 receptors in the amygdalar cholecystokinin glutamatergic afferents to nucleus accumbens modulate depressive-like behavior.

Major depressive disorder is a devastating psychiatric disease that afflicts up to 17% of the world's population. Postmortem brain analyses and imaging studies of patients with depression have implicated basal lateral amygdala (BLA) dysfunction in the pathophysiology of depression. However, the circuit and molecular mechanisms through which BLA neurons modulate depressive behavior are largely uncharacterized. Here, in mice, we identified that BLA cholecystokinin (CCK) glutamatergic neurons mediated negative reinforcement via D2 medium spiny neurons (MSNs) in the nucleus accumbens (NAc) and that chronic social defeat selectively potentiated excitatory transmission of the CCKBLA-D2NAc circuit in susceptible mice via reduction of presynaptic cannabinoid type-1 receptor (CB1R). Knockdown of CB1R in the CCKBLA-D2NAc circuit elevated synaptic activity and promoted stress susceptibility. Notably, selective inhibition of the CCKBLA-D2NAc circuit or administration of synthetic cannabinoids in the NAc was sufficient to produce antidepressant-like effects. Overall, our studies reveal the circuit and molecular mechanisms of depression.

Paracrine-like excitation of low-threshold mechanoceptive C-fibers innervating rat hairy skin is mediated by substance P via NK-1 receptors.

We reported previously that C-fibers innervating rat skin can be excited by short trains of electrical shocks ('tetanus') applied to neighboring nerves. Since these nerves were disconnected from the CNS, the cross-talk is located peripherally. Here we tested if low-threshold mechanoceptive (LTM) C-fibers can be excited by this cross-talk and if this process is mediated by substance P (SP) via neurokinin-1 (NK-1) receptors. In urethane anesthetized rats we found that 80% (56/71) of LTM C-fibers, recorded in the lateral cutaneous branch of the dorsal ramus (CBDR) of T10 spinal nerve, were excited by a 10s, 20 Hz tetanus of the T9 CBDR. Compared to the spontaneous pre-tetanic firing frequency of 1.62+/-0.40 impulses/30s, the frequency significantly increased to 3.74+/-0.99, 3.17+/-0.69 and 2.92+/-0.63 impulses/30s, at 30, 60 and 90 s after the tetanus, respectively, and declined to the baseline frequency thereafter. When injected into their receptive fields, SP mimicked the tetanically induced increase of firing rate, whereas the NK-1 receptor antagonist WIN 51708 blocked the excitation in most fibers. The excitation was significantly diminished in adult rats that were neonatally treated with capsaicin, a treatment that destroys most SP-expressing afferent fibers. Thus, we conclude that peptidergic primary afferents are functionally linked with adjacent LTM C-fibers in a non-synaptic, paracrine-like signaling pathway via SP and NK-1 receptors, and perhaps also other agents as well. We propose that this cross-talk has evolved as a mechanism regulating the mechanoceptive characteristics of LTM C-fibers, presumably contributing to pain sensation elicited by tactile stimuli ('allodynia').

[Integrating genetic and gene expression data: methods and applications of eQTL mapping].

The availability of high-throughput genotyping technologies and microarray assays has allowed researchers to investigate genetic variations that influence levels of gene expression. Expression Quantitative Trait Locus (eQTL) mapping methods have been used to identify the genetic basis of gene expression. Similar to traditional QTL studies, the main goal of eQTL is to identify the genomic locations to which the expression traits are linked. Although microarrays provide the expression data of thousands of transcripts, standard QTL mapping methods, which are able to handle at most tens of traits, cannot be applied directly. As a result, it is necessary to consider the statistical principles involved in the design and analysis of these experiments. In this paper, we reviewed individual selection, experimental design of microarray, normalization of gene expression data, mapping methods, and explaining of results and proposed potential methodological problems for such analyses. Finally, we discussed the applications of this integrative genomic approach to estimate heritability of transcripts, identify candidate genes, construct gene networks, and understand interactions between genes, genes and environments.

[Apoptosis of myeloid leukemia cell line HL60 induced by Bortezomib, a proteasome inhibitor].

OBJECTIVE: To explore the mechanism of apoptosis of myeloid leukemia cells induced by Bortezomib, a proteasome inhibitor. METHODS: Human acute myeloid leukemia cells of the line HL60 were cultured and treated with Bortezomib of the concentrations of 0, 10, 20, 30, and 40 micromol/L for 24 hours. MTT assay and flow cytometry were used to detect the proliferation inhibition and apoptosis. Hoechst 33342 staining was used to observe the morphology of the cells. Western blotting was used to detect the protein expression of Bcl-2, Caspase-9, Caspase-3, and poly ADP-ribose polymerase (PARP). RESULTS: Bortezomib could induce HL60 cell apoptosis dose- and time-dependently. After treated for 24 hours by 30 nmol/L Bortezomib the HL60 cells' proliferation was significantly inhibited, the inhibition rate was 76%, and the cell nuclei became progressively pyknotic and were extensively fragmented. FCM showed apoptosis peaks 24 hours after treatment of Bortezomib of the concentrations of were 10 and 20 nmol/L, the apoptosis rate was 62.6%. Bcl-2 protein expression was down-regulated and the protein expressions of Caspase-9, Caspase-3, and PARP were all up-regulated. CONCLUSION: The mechanism of Bortezomib to induce apoptosis of myeloid leukemia cells is associated with down-regulation of Bcl-2 protein expression and cleaved activation of Caspase-9, Caspase-3 and PARP proteins.

[The mixed major gene plus polygenes inheritance for female fertility in wheat (Triticum aestivum L.)].

Three sets of data for the P1, P2, F1, and F2 populations derived from three crosses between the normal fertility wheat (Triticum aestivum L.) cultivars with different ecotypes and the female sterile line (XND126) were used to investigate the inheritance of female fertility in wheat using mixed major gene plus polygenes inheritance model in 2005 and 2006. The results from the joint segregation analysis of the four generations showed that female fertility in wheat is controlled by two major genes plus polygenes, and the interaction between the two major genes is also detected.

[Comparative analysis of genetic diversity revealed by genomic-sSR, EST-SSR and pedigree in wheat (Triticum asetivum L.)].

Two SSR molecular markers, genomic-SSR and EST (expressed sequence tagged)-SSR, were used to measure the genetic diversity among 18 accessions of common wheat with known pedigrees, which were collected from winter wheat production region in Northern China. In addtion, the genetic diversity revealed by pedigree, EST-SSR and genomic-SSR was also compared. The results showed that the average number of alleles per genomic-SSR locus is 3.34, which is higher than that of EST-SSR (2.31), indicating that genomic-SSR is more polymorphic than EST-SSR. Genomic-SSR and EST-SSR were used to calculate the genetic distance (GD) in different materials. The mean GD value of EST-SSR for the 18 wheat genotypes is 0.3996,which is lower than that of genomic-SSR (0.5458). At the locus level, the mean GD calculated using pedigree information is higher (0. 9716) than that of genomic-SSR and EST-SSR markers (0.5458 and 0.3996). Therefore, although polymorphism of EST-SSR is low as compared to genomic SSR,it provides a more accurate evaluation of genetic relationship, especially when accessions are very closely related in pedigree. The strategy for improving the genetic diversity of wheat was also discussed.

High-density genetic linkage map construction and QTL mapping of grain shape and size in the wheat population Yanda1817 × Beinong6.

High-density genetic linkage maps are necessary for precisely mapping quantitative trait loci (QTLs) controlling grain shape and size in wheat. By applying the Infinium iSelect 9K SNP assay, we have constructed a high-density genetic linkage map with 269 F 8 recombinant inbred lines (RILs) developed between a Chinese cornerstone wheat breeding parental line Yanda1817 and a high-yielding line Beinong6. The map contains 2431 SNPs and 128 SSR & EST-SSR markers in a total coverage of 3213.2 cM with an average interval of 1.26 cM per marker. Eighty-eight QTLs for thousand-grain weight (TGW), grain length (GL), grain width (GW) and grain thickness (GT) were detected in nine ecological environments (Beijing, Shijiazhuang and Kaifeng) during five years between 2010-2014 by inclusive composite interval mapping (ICIM) (LOD ≥ 2.5). Among which, 17 QTLs for TGW were mapped on chromosomes 1A, 1B, 2A, 2B, 3A, 3B, 3D, 4A, 4D, 5A, 5B and 6B with phenotypic variations ranging from 2.62% to 12.08%. Four stable QTLs for TGW could be detected in five and seven environments, respectively. Thirty-two QTLs for GL were mapped on chromosomes 1B, 1D, 2A, 2B, 2D, 3B, 3D, 4A, 4B, 4D, 5A, 5B, 6B, 7A and 7B, with phenotypic variations ranging from 2.62% to 44.39%. QGl.cau-2A.2 can be detected in all the environments with the largest phenotypic variations, indicating that it is a major and stable QTL. For GW, 12 QTLs were identified with phenotypic variations range from 3.69% to 12.30%. We found 27 QTLs for GT with phenotypic variations ranged from 2.55% to 36.42%. In particular, QTL QGt.cau-5A.1 with phenotypic variations of 6.82-23.59% was detected in all the nine environments. Moreover, pleiotropic effects were detected for several QTL loci responsible for grain shape and size that could serve as target regions for fine mapping and marker assisted selection in wheat breeding programs.

[Genetic diversity of D-genome revealed by SSR markers in synthesized hexaploid wheat introduced from CIMMYT].

Simple sequence repeat (SSR) molecular marker was used to measure the genetic diversity of D-genome in 26 synthesized hexaploid wheat (AABBDD) introduced from CIMMYT. Twenty-three D-genome specific SSR primers were selected for PCR amplification, among which 22 primers can detect polymorphism. A total of 92 alleles were identified at 23 loci using the above SSR primers, with an average of 4 alleles per locus. The 92 alleles were used to calculate Nei's similarity index (GS) and the genetic distance (GD). It was also found that the mean genetic distance between 26 synthesized hexaploid wheat was 0.4955, which was obviously high. From the above results, it can be indicated that the genetic variation of D-genome in synthesized hexaploid wheat was abundant and could be used to improve the genetic diversity in wheat breeding. Interestingly, synthesized hexaploid wheat 17 and 18 shared the same D-genome donor, but three of 23 detected SSR loci were polymorphic between the two materials. Therefore, during the period of allopolyploidization, there was genetic differentiation in repeat region of donor genome.

[Genetic diversity revealed by ISSR molecular marker in common wheat, spelt, compactum and progeny of recurrent selection].

It is important to estimate the genetic diversity between the parents for improving the heterosis of hybrid wheat. In this study, ISSR(inter-simple sequence repeat) marker was used to measure the genetic diversity within and among common wheat (Triticum aestivum L.), spelt (Triticum spelta L.), compactum (Triticum compactum Host.) and progeny of foreign wheat-based recurrent selection, and the possibility of establishing the new heterotic group was also assessed. Forty seven genotypes were used for ISSR analysis, which included 14 common wheat, 10 spelt wheat, 11 compactum and 12 progeny of recurrent selection. Eleven of 33 ISSR primers that can produce distinguishable bands were selected for PCR amplification. A total of 238 bands were amplified, among which 207 (87%) bands were polymorphic. The polymorphic bands amplified by each primer ranged from 11 to 38, with an averaged of 18.8. The percentage of polymorphic band (80.3%) in common wheat was higher than that in progeny of recurrent selection (78.7%), spelt (75.0%) and compactum (74.9%). The 238 polymorphic products were used to calculate Nei's similarity index (GS) and the genetic distance (GD). It was found that the mean genetic distance between different wheat types (0.3115-0.3442) was obviously higher than that within common wheat (0.2743), spelt (0.2351), compactum (0.2622). In addition, progeny of recurrent selection also showed much higher genetic distance with other three wheat types (0.3217, 0.3256, 0.3198). The cluster analysis was performed based on the genetic distance (GD) matrix by using UPGMA method. Common wheat, spelt, compactum and progeny of recurrent selection were classified into four different groups. In this study, ISSR marker was firstly used to assess genetic diversity among common wheat, spelt, compactum and progeny of recurrent selection, and can differentiate the wheat cultivars (lines) that selected from the same cross combination. It was concluded that spelt, compactum and progeny of recurrent selection can be used to diversify the genetic basis for hybrid wheat breeding and improve heterosis. It is possible to establish the wheat heterotic group by ISSR marker.

Relationship between differences of gene expression in early developing seeds of hybrid versus parents and heterosis in wheat.

In order to understand molecular basis of heterosis, mRNA differential display was used to analyze the differences in gene expression between seeds of 18 reciprocal hybrids and their 6 parents at 6th day after pollination. The relationship between gene expression patterns and heterosis was determined. Only bands that can be repeated in duplicate PCR were used for analysis so as to reduce false positive bands. Among the total of 2,025 bands displayed, 1,386 bands (68.43%) were reproducible. Eight patterns (fifteen kinds) of gene expression were observed, which include: (1) bands occurring in only one parent (two kinds); (2) bands observed in both hybrids and one parent (two kinds); (3) bands detected in parents and one hybrid (two kinds); (4) bands displayed in only one hybrid (two kinds); (5) bands revealed in one hybrid and its corresponding female (two kinds) or male (two kinds) parent; (6) bands visualized in only both hybrids (one kind); (7) bands occurring in only parents (one kind); (8) bands observed in parents and both hybrids (one kind). Our results indicate that differences of gene expression between hybrids and their parents are very obvious. The percentages of bands observed in only both hybrids and in only both parents are lower. The analysis shows that bands observed in parents and both hybrids are not correlated with all nine agronomic traits, which indicates differentially expressed genes are mainly responsible for the observed heterosis. At least one pattern of differential gene expression is significantly correlated with nine agronomic traits. Silenced bands in hybrid (which include bands occurring in only one parent, bands detected in both parents and one hybrid and bands in only parents) and bands present in one hybrid and its corresponding female or male parent are likely to play important roles in heterosis. These results suggest that early seed development could be closely related to heterosis.

[Changes in mechano-receptive properties of Abeta-fiber induced by antidromical electrical stimulation of the cutaneous nerve from adjacent spinal segment].

The purpose of the present study was to observe whether primary afferent Abeta-fiber is involved in the information transmission between peripheral terminals of adjacent dermatomes. The dorsal cutaneous nerve branches of spinal nerves from T(8) to T(12) segments were cut proximally. One peripheral stump end of the cut nerves was dissected into a few filaments for the examination of mechanoreceptive properties of single Abeta-fibers and their discharges were observed while the other end was stimulated antidromically. Fifty Abeta-units were recorded in forty-two intact rats. After an electrical stimulation (0.45 mA, 0.1 ms, 20 Hz, for 10 s) was delivered to the stimulated nerve, the size of the receptive field of 60.6% (n=33) Abeta-fibers extended. The mean area of receptive fields of all examined units enlarged from 8.94+/-6.51 mm(2) to 20.34+/-16.17 mm(2) (P<0.01) and the shapes of the receptive fields of 81.8% (n=20) units changed from a dot, round or ellipse with its long axis in parallel with the longitudinal axis of the body to an oblique ellipse with the longitudinal axis of the body. The mechanoreceptive threshold of 68.0% (n=50) units decreased with a reduction in mean threshold from 2.37+/-1.24 to 2.29+/-1.24 mN (P<0.05). The duration of these changes in mechano-receptive properties increased from 52.23+/-9.27 to 56.93+/-15.76 min. Meanwhile, increasing discharge was found in 50.0% (n=50) units but lasted only for 1.52+/-0.46 min. The changes in mechanoreceptive properties appeared simultaneously with discharge changes but had longer duration than that of discharge change (P<0.01). Discharges changes usually appeared in those units with the changes in mechanoreceptive properties following an antidromical electrical stimulation of adjacent spinal segment. These results suggest that low-threshold mechanoreceptive Abeta-fibers are affected by antidromical electrical stimulation of the cutaneous nerve from an adjacent spinal segment, indicating that information transmission occurs between the two endings of peripheral afferent nerves from adjacent spinal segments without any involvement of the central nervous system, and that Abeta-fibers are involved in the process of information transmission between peripheral terminals from adjacent spinal segments.

A wheat lipid transfer protein 3 could enhance the basal thermotolerance and oxidative stress resistance of Arabidopsis.

Wheat (Triticum aestivum L.) is one of the major grain crops, and heat stress adversely affects wheat production in many regions of the world. Previously, we found a heat-responsive gene named Lipid Transfer Protein 3 (TaLTP3) in wheat. TaLTP3 was deduced to be regulated by cold, ABA, MeJA, Auxin and oxidative stress according to cis-acting motifs in its promoter sequences. In this study, we show that TaLTP3 is responsive to prolonged water deficit, salt or ABA treatment in wheat seedlings. Also, TaLTP3 accumulation was observed after the plant suffered from heat stress both at the seedling and the grain-filling stages. TaLTP3 protein was localized in the cell membrane and cytoplasm of tobacco epidermal cells. Overexpression of TaLTP3 in yeast imparted tolerance to heat stress compared to cells expressing the vector alone. Most importantly, transgenic Arabidopsis plants engineered to overexpress TaLTP3 showed higher thermotolerance than control plants at the seedling stage. Further investigation indicated that transgenic lines decreased H2O2 accumulation and membrane injury under heat stress. Taken together, our results demonstrate that TaLTP3 confers heat stress tolerance possibly through reactive oxygen species (ROS) scavenging.

[In vitro effect of bortezomib alone or in combination with harringtonine or arsenic trioxide on proliferation and apoptosis of multidrug resistant leukemia cells].

OBJECTIVE: To investigate the effect of bortezomib alone or combined with harringtonine (HT) or arsenic trioxide (As2O3) on the proliferation capacity and apoptosis of HL-60/ADM cell line and fresh cells from refractory/relapse acute leukemia patients. METHODS: HL-60/ADM cells or refractory/relapse leukemia cells were incubated with bortezomib at different doses alone and in combination with HT or As2O3. The proliferation capacity was observed by MTT assay, cell apoptosis by fluorescence microscopy and flow cytometry. Intracellular concentration of daunorubicin (DNR) was determined by flow cytometry. RESULTS: In bortezomib-treated HL-60/ADM cells, the proliferation inhibition rate and apoptotic cells increased in a time- and dose-dependent manner. 40 nmol/L bortezomib could maximally inhibit the proliferation of HL-60/ADM cells at 48 hours. 15 micromol/L As2O3 or 752 nmol/L HT combined with different doses of bortezomib could inhibit proliferation and induce apoptosis of HL-60/ADM cells. The As2O3 plus bortezomib or HT plus bortezomib showed a greater anticancer efficacy than either of the drugs alone (P < 0.05, P < 0.01). Bortezomib (10 nmol/L) could markedly enhance the intracellular accumulation of DNR in HL-60/ADM cells (P < 0.05). CONCLUSIONS: Bortezomib can inhibit proliferation and induce apoptosis of HL-60/ADM cells and fresh refractory/relapse acute leukemia cells, especially combined with HT or As2O3.

[The effect of Haynaldia villosa v chromosome on the mitochondrial proteome of wheat-H. villosa chromosome substitution line and translocation line].

Wheat-Haynaldia villosa chromosome substitution line (6A/6V) and translocation lines (6DL/6VS, 6AL/6VS) were obtained through hybridization of H. villosa with powdery mildew susceptible cultivated wheat. Substitution line and translocation lines contain V chromosome or the chromosome short arm (VS) of H. villosa. They are resistant to powdery mildew. In this study, mitochondrial proteome changes were analyzed by using substitution line (6A/6V), translocation line (6DL/6VS) as experimental materials in order to studying the effects of V chromosome on the mitochondrial proteome and related to powdery mildew resistance. The results indicated that 16 new mitochondrial protein spots (spot1, 22kDa/PI8.5; spot2, 31 kDa/PI 7.5; spot3, 28 kDa/PI 7.0; spot4, 31 kDa/PI 6.5; spot5, 40 kDa/PI 7.5; spot6, 40 kDa/PI 7.4; spot7, 80 kDa/PI 8.4; spot8, 50 kDa/PI 7.5; spot9, 60 kDa/PI 7.3; spot10, 65 kDa/PI 6.6; spot11, 65 kDa/PI 6.6; spot12, 73 kDa/PI 7.5; spot13, 73 kDa/PI 7.7; spot14, 46 kDa/PI 7.4; spot15, 46 kDa/PI 7.3; spot16, 38 kDa/PI 6.3) were produced and 7 mitochondrial protein spots (spot1, 40 kDa/PI 7.5; spot2, 43 kDa/PI 7.6; spot3, 48 kDa/PI 7.5; spot4, 42 kDa/PI 8.0; spot5, 43 kDa/PI 7.5; spot6, 32 kDa/PI 4.8; spot7, 40 kDa/PI 5.5) were absent in substitution line, 7 new mitochondrial protein spots (spotl, 43 kDa/PI 6.3; spot2, 60 kDa/PI 6.5; spot3, 60 kDa/PI 6.4; spot4, 65 kDa/PI 7.5; spot5, 55 kDa/PI 8.2; spot6, 31 kDa/PI 8.0; spot7, 43 kDa/PI 8.0) were produced and 6 mitochondrial protein spots (spot1', 66 kDa/PI 8.3; spot2', 58 kDa/PI 8.5; spot3', 36 kDa/PI 7.0; spot4', 48 kDa/PI 7.7; spot5', 48 kDa/PI 6.8; spot6', 43 kDa/PI 6.2) were absent in translocation line. These experimental results suggest that V chromosome or VS of H. villosa can obviously lead mitochondrial proteome changed. These changes may be associated with resistant to powdery mildew of substitution line and translocation line.

Screening and identification of the AFLP markers linked to a new powdery mildew resistance gene in wheat cultivar Brock.

Wheat cultivar Jing 411 which is susceptible to powdery mildew, wheat cultivar Brock and near isogenic lines (NILs) of Jing411, which are resistant to powdery mildew were analysized for polymorphisms using 225 pairs of AFLP primers Only two pairs of primers Pst+GAC/Mse+ TCT (P1) and Pst+AGC/Mse+ACC (P2) stably produced polymorphic bands between the resistant and susceptible plants. Two specific fragments were obtained. By cloning and sequencing these two specific fragments, it was showed that the specific fragment amplified by primer P1 had 268bp, and the fragment amplified by P2 had 227bp. They were named AFLP marker P1(268) and P2(227) respectively. Linkage analysis of these two markers revealed that the polymorphism existed in a 106 F2 segregating population. These two markers closely linked to a powdery mildew resistance gene in wheat cultivar Brock, linkage distance were 3.6 and 1.9 cM respectively. These two markers will be useful for marker-assisted selection and gene pyramiding in wheat resistance breeding.